The conserved coiled-coil protein CCCP1 and the endosomal complex EARP regulate dense-core vesicle cargo sorting
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The regulated release of peptide hormones (e.g. insulin), neuropeptides, and monoamines depends on their being packaged into dense-core vesicles (DCVs). How these vesicles are made and how their cargo is properly selected is not well understood, especially at the molecular level. DCVs are generated at the trans-Golgi network (TGN) and go through maturation steps including peptide processing and removal of improperly loaded cargo. Screens in C. elegans have identified several conserved molecules that are important for DCV function. These include the small G protein RAB2, its effector the conserved coiled-coil protein 1 (CCCP1), the endosome-associated recycling complex (EARP), and the EARP interacting protein 1 (EIPR1). All these proteins function in C. elegans neurons in the same genetic pathway. In mutants of these proteins, DCVs are generated normally but they contain reduced levels of cargo suggesting a role in cargo sorting. To determine whether CCCP1 and EIPR1 are important for the formation of DCVs in mammalian endocrine cells, we generated Cccp1KO and Eipr1KO insulin-secreting pancreatic beta 832/13 cells. We have observed that the KO cells have reduced insulin secretion and that insulin is retained near or at the TGN suggesting that both proteins are required for sorting insulin into DCVs. In a proximity labelling BioID screen, I found that CCCP1 is in close proximity to the transmembrane protein carboxypeptidase D (CPD). CPD localizes to the TGN and to immature DCVs (iDCVs), but gets removed from iDCVs during maturation. Interestingly, I have shown that in Cccp1KO and in Eipr1KO cells, CPD is missorted and remains in mature DCVs. In subcellular localization studies, I found that both CCCP1 and EIPR1/EARP partially colocalize with TGN and iDCV markers, while a second pool of EARP localizes to recycling endosomes. By super-resolution microscopy, I observed that CCCP1 forms circles (~200 nm diameter) that surround proinsulin; suggesting that CCCP1 localizes around immature DCV membranes, around proinsulin positive TGN subdomains, or to both. Together, these localization and functional studies suggest that CCCP1 and EIPR1 are novel regulators of DCV cargo sorting in neurons and endocrine cells. The data is consistent with a model in which CCCP1 and EIPR1 control insulin sorting into DCVs at the TGN and remove cargo not destined to the regulated secretory pathway in a post-Golgi step. To gain insights into the function of CCCP1, I have performed a detailed structure-function study of CCCP1. I have shown that its C-terminal coiled-coil CC3 domain is necessary and sufficient for its localization in vivo, while its middle coiled-coil domain CC2 is required for recruiting EARP complex (likely EARP positive membrane compartment). Finally, in biophysical studies of CCCP1, I have found that the protein has features reminiscent of golgins, a family of membrane tethers. Together, my findings are consistent with a speculative model in which a membrane tethering step between TGN/iDCVs membranes and EARP positive membranes might be required for cargo sorting to DCVs.
- Biological chemistry